Use Of Modulators Of CCR5 In The Treatment Of Cancer And Cancer Metastasis

ABSTRACT

This disclosure is directed, in part, to a method of determining whether a subject having cancer is at risk for developing metastasis of the cancer. In one embodiment, the method comprises (a) obtaining a biological sample from the subject having cancer; (b) determining CCR5 expression level and/or expression level of at least one of CCR5 ligands in the biological sample; and (c) if the expression level of CCR5 and/or of at least one of CCR5 ligands determined in step (b) is increased compared to CCR5 expression level and/or expression level of at least one of CCR5 ligands in a control sample, then the subject is identified as likely at risk for developing metastasis of the cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/893,791, filed May 14, 2013, which claims the benefit of U.S.Provisional Patent Application Nos. 61/646,586, filed May 14, 2012, andentitled “Use Of Modulators Of CCR5 In The Treatment Of Cancer AndCancer Metastasis”; and 61/646,593, filed May 14, 2012, and entitled“Use Of Modulators Of CCR5 In The Treatment Of Cancer And CancerMetastasis”, each of which are incorporated by reference herein in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was supported in part by PASPA-UNAM (M.A.V-V.), NIHgrants R01CA070896, R01CA075503, R01CA132115, R01CA107382, R01CA086072(R.G.P.), R01CA120876 (M.P.L), the Kimmel Cancer Center NIH CancerCenter Core grant P30CA056036 (R.G.P.), generous grants from the Dr.Ralph and Marian C. Falk Medical Research Trust and the Margaret Q.Landenberger Research Foundation, and a grant from PennsylvaniaDepartment of Health (R.G.P.). Accordingly, the United States governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This disclosure is directed, in part, to a method of determining whethera subject having cancer is at risk for developing metastasis of thecancer and a method of blocking cancer methastasis.

BACKGROUND OF THE INVENTION

Breast cancer causes the death of 40,000 women in the USA and 410,000women in the world annually.¹ Despite advances in the treatment of thedisease, 20% to 30% of patients with early breast cancers willexperience relapse with distant metastatic disease.² In those patients,metastasis is the main cause of death. Patients with basal tumors haveincreased risk of metastasis and lower survival rate.³′ ⁴ Kennecke etal. studied 3,726 breast cancer patients and reported that the basaltumors have higher frequencies of metastases and reduced time fromidentification of metastases to death compared to that of patients withluminal A or B tumors.⁴ The absence of AR, ER, and HER-2 commonly foundin basal breast tumors⁵ means that they are unlikely to respond tohormone therapies or HER-2 targeted therapies. Currently, chemotherapy,radiation, and surgery are the only choices for patients with basalbreast cancers, but all demonstrate poor outcomes.⁶ The need for aspecific targeted therapy for basal breast cancer remains urgent.

BRIEF SUMMARY OF THE INVENTION

Certain aspects of embodiments disclosed herein by way of example aresummarized below. It should be understood that these aspects arepresented merely to provide the reader with a brief summary of certainforms an invention disclosed and/or claimed herein might take and thatthese aspects are not intended to limit the scope of any inventiondisclosed and/or claimed herein. Indeed, any invention disclosed and/orclaimed herein may encompass a variety of aspects that may not be setforth below.

The present disclosure generally relates to various methods, includingmethods of determining whether a subject has cancer or is at risk fordeveloping cancer and/or is at risk for developing cancer metastasis. Insome embodiments, the methods of the present invention include methodsof treating, preventing, or managing a neoplasm or a cancer metastasisin a patient. In some embodiments, the methods of the present inventioninclude in vivo methods for down regulating CCR5 expression in a set ofone or more prostate cancer cell lines derived from transduction ofmurine epithelial or prostate epithelial cells and transformed by atleast one oncogene selected from the group consisting of NeuT, Ha-Ras,and c-Src. In some exemplary embodiments drugs that target the HIVreceptor CCRS, which the virus uses to enter and infect host cells, areused to prevent migration and spread of cancer cells from their primarytissue to secondary sites in the body of the patient. In one embodiment,a drug that targets the HIV receptor CCR5 may be used as an adjuvanttherapy, or adjuvant care, wherein the drug is administered to thepatient in addition to a primary, main or initial treatment for cancer.In some embodiments, such adjuvant therapy may be administeredconcomitantly or concurrently as other therapies for cancer. In oneembodiment, such adjuvant therapy may be given concurrently as otheradjuvant therapies or following other adjuvant therapies.

In some embodiments, when CCR5 receptor antagonists are used as adjuvanttherapy they improve prognosis in cancer patients. In some embodiments,where a plurality of anticancer drugs are used in combination for cancertherapy, a CCR5 receptor antagonist may be included as an adjuvanttherapy in order to improve the therapeutic effect by blockingmetastasis of the cancer being treated, thereby contributing toimproving clinical outcomes of the cancer therapy.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as other features, aspects, andadvantages of the present invention and the following detaileddescription of embodiments of the invention, will be better understoodwhen read in conjunction with the claims and the appended drawings of anexemplary embodiments, wherein:

FIG. 1A illustrates a heatmap of the expression of CCL5 and its receptorCCR5 in samples from patients with breast cancer divided by molecularsubtype (luminal A, luminal B, basal, normal-like, and Her-2 ) based ontheir gene expression pattern;

FIG. 1B includes plots 1B1 through 1B5, which illustrates scatter plotsand correlation analysis (Student t test) of the expression of CCL5 andCCR5 among the breast cancer molecular subtypes of luminal A, luminal B,basal, normal-like, and Her-2;

FIG. 1C illustrates quantification of the proportions of the breastcancer samples overexpressing CCL5 and CCR5, fraction of the barsrepresenting upper right quadrants of the scatter plots shown in FigureB;

FIG. 1D illustrates metastasis-free Kaplan-Meier plots and log-rankanalysis for the different molecular subtypes of breast cancer in theanalyzed database, which is described in Materials and Methods sectionof this of this application;

FIG. 2A illustrates flow cytometric histograms of the CCR5 expression inMDA-MB-231 breast cancer cells identified a subpopulation of CCR5⁺cells;

FIG. 2B includes plot 2B1 and illustrates induction of calcium signalingin cells loaded with Fluo-4-AM before the sequential addition of CCL5(60 μg/mL) and FBS (5%);

FIG. 2C illustrates 3D invasion into collagen gels by breast cancer celllines, using CCL5 (15 μg/mL) as chemoattractant;

FIG. 2D illustrates mean distances of invasion±SEM from 3 independentexperiments;

FIG. 2E 3D invasion assays for MCF-10A cells and MCF-10A-NeuT, -Ras, and-Src derivatives showing that CCL5 -induced invasion is activated byoncogenic transformation;

FIG. 2F illustrates quantification (F, mean±SEM, n=3) 3D invasion assaysfor MCF-10A cells and MCF-10A-NeuT, -Ras, and -Src derivatives showingthat CCL5 -induced invasion is activated by oncogenic transformationshown in FIG. 2E;

FIG. 2G illustrates CCR5+ and CCR5− subpopulations from SUM-159 cellline that were isolated by FACS and their respected invasion intocollagen gels evaluated using fetal bovine serum (FBS) aschemoattractant;

FIG. 2H illustrates quantification of the invasion of CCR5+ and CCR5−subpopulations into collagen as mean±SEM of the two independentexperiments shown in FIG. 2G;

FIG. 3A includes image 3A1, image 3A2, graph 3A3 and graph 3A4 andillustrates intensity versus time analysis of Fluo-4 AM-loadedMDA-MB-231 cells treated with the CCR5 antagonists maraviroc orvicriviroc (100 nmol/L) for 30 minutes before the addition of CCL5 (60m/mL);

FIG. 3B illustrates a comparison of the fraction of cells with increasedfluorescence intensity upon addition of CCL5;

FIG. 3C includes image 3C1, image 3C2, graph 3C3 and graph 3C4 and showsCCL5-induced calcium signaling blocked by CCR5 antagonists in Hs578Tcells;

FIG. 3D illustrates quantification (mean±SEM) of 3 to 4 independentexperiments shown in FIGS. 3A-3C;

FIG. 4A illustrates 3D reconstruction of FBS-induced invasion intocollagen gels by Hs578T breast cancer cells in presence of CCR5antagonists (100 nmol/L);

FIG. 4B illustrates quantifications (mean±SEM, n=3) and analysis(Bonferroni t test) of the FBS-induced invasion into collagen gels byHs578T breast cancer cells shown in FIG. 4A;

FIG. 4C illustrates 3D reconstruction of FBS-induced invasion intocollagen gels by SUM-159 breast cancer cells in presence of CCR5antagonists (100 nmol/L);

FIG. 4D illustrates quantifications (mean±SEM, n=3) and analysis(Bonferroni t test) of the FBS-induced invasion into collagen gels bySUM-159 breast cancer cells shown in FIG. 4A;

FIG. 5A shows exemplary in vivo bioluminescent images (BLIs) of vehicle-or maraviroc-treated (8 mg/kg every 12 hours) of nonobese diabetic(NOD)/severe combined immunodeficiency (SCID) mice (hereinafterabbreviated as NOD/SCID mice);

FIG. 5B shows quantification (mean±SEM, n=6) of in vivo bioluminescentimages (BLIs) in the control (vehicle-treated NOD/SCID mice, redline/squares) and treated groups (maraviroc-treated NOD/SCID mice, blueline/triangles);

FIG. 5C includes images 5C1 and 5C2 and shows the presence of pulmonarytumors and the differences between treatments corroborated by ex vivoimaging (left) and India ink staining (right);

FIG. 5D showing the fraction of mice with metastatic tumors beingsignificantly larger in the control group (P<0.0001, Fisher exact test);

FIG. 5E showing histological analysis (hematoxylin and eosin staining,×100) of the area covered by metastatic tumors in lung slides;

FIG. 5F shows quantification of the area covered by metastatic tumors inlung slides shown in FIG. 5E;

FIG. 6A, effect of CCR5 antagonist on breast cancer cell viability.MDA-MB-231 cells were exposed to increasing concentrations of maraviroc(inverted triangles) or vicriviroc (squares) for 48 hours and the cellviability was evaluated by MTT assay.

FIG. 6B illustrates flow cytometric analysis showing CCR5 expression inMDA-MB-231 cells stably transfected with pcDNA3.1⁺/Zeo⁺(MDA.Vector) orhuman CCR5 cloned into pCDNA3⁺Zeo⁺ (MDA.CCR5).

FIG. 6C illustrates a comparison of in vitro proliferation rates ofMDA-MB-231 in MDA.Vector and MDA.CCRS;

FIG. 6D illustrates evaluation of the in vivo effect of maraviroc ongrowth of established metastasis in mice, wherein treatment of mice wasinitiated 10 days after injection of MDA.pFULG cells as illustrated, andin vivo bioluminescence imaging (BLI) of the mice for evaluating theefficacy of the treatment was carried out in days: 0, 10, 17, 24, 31 and38;

FIG. 6E illustrates quantification (mean±SEM, n=5) of in vivo BLI in thecontrol (red/squares) and treated groups (blue/triangles) showed nodifferences in the growth rate;

FIG. 6F illustrates an schema of the experimental design used toevaluate CCR5 role in lung colonization by MDA-MB-231 cells;

FIG. 6G illustrates exemplary confocal images of eGFP⁺ cells in lungs ofmice 24 hours after injection of the mice with MDA.pFULG cells, whereincells expressing eGFP were counted in 3 random fields of 2 differenthistologic sections (separated 700 μm from each other) per mouse (n=5mice per group);

FIG. 6H illustrates quantification (H) of the number of eGFP⁺ cells inlungs of mice 24 hours after injection of the mice with MDA.pFULG cells,wherein cells expressing eGFP were counted in 3 random fields of 2different histologic sections (separated 700 μm from each other) permouse (n=5 mice per group);

FIG. 7A illustrates GSEA analysis using KEGG and GO of tumor samples todetermine the gene expression signaling pathway associated withenrichment of CCR5 and CCL5, wherein genes in the examined breast cancerdataset are ranked by a signal-to-noise metric representing theirdifferential expression in highest 2.5% CCL5/CCR5 -expressing samplesversus lowest (2.5%) CCL5/CCR5 expressing samples (N=54), depicted witha color gradient, where red indicates positive correlation withCCL5/CCR5 expression and blue represents negative correlation withCCL5/CCR5 expression;

FIG. 7B includes scatter plots 7B1 through 7B5 and illustrates scatterplots and correlation analysis using students' t-tests for theexpression of CCR1 and CCR5 among the breast cancer molecular subtypesfrom a set of 2250 human breast cancer data shown in FIG. 1A;

FIG. 7C includes scatter plots 7C1 through 7C5 and shows scatter plotsand correlation analysis for the expression of CCR3 and CCL5 among thebreast cancer molecular subtypes from a set of 2250 human breast cancerdata shown in FIG. 1A;

FIG. 7D includes Kaplan Meier curves 7D1 and 7D2 which are Kaplan Meiercurves for patients with breast cancer samples enriched for the highestlevel of CCR5 (up of 50%) versus the lower level CCR5 expression (lower50%);

FIG. 8A illustrates heat map of CCL5 expression and its receptors CCR5,CCR1 and CCR3 in healthy population.

FIG. 8B illustrates heat map representing cross correlation of CCL5expression and its receptors CCR5, CCR1 and CCR3 amongst healthypopulation;

FIG. 8C illustrates heat map representing cross correlation of CCL5expression and its receptors CCR5, CCR1 and CCR3 amongst breast cancerpatients derived from a collection of 2550 breast cancers;

FIG. 9A illustrates flow cytometry plot of the CCR5 expression in HS578Tcells stably transfected with allophycocyanin (APC)-labeled antibody toCCR5;

FIG. 9B includes plot 9B1 and illustrates induction of calcium signalingin HS578T cells loaded with Fluo-4-AM before the sequential addition ofCCL5 (60 μg/mL) and fetal bovine serum (FBS) (5%), wherein RFIrepresents relative fluorescence intensities. The red traces are ofcells that did not respond to the addition of CCL5 ligand—but do respondto FBS (fetal bovine serum), the green line is the trace for the cellsthat do respond to the additionaof CCL5, and also respond to FBS. Thisdata tells us that the MDA-MB-231 cells are a population of cells inwhich some cells have the receptor and respond to CCL5− and some cellsthat do not have the CCR5 receptor and do not respond to its ligandCCL5;

FIG. 9C illustrates flow cytometry plot of the CCR5 expression in SUM159cells stably transfected with allophycocyanin (APC)-labeled antibody toCCR5.

FIG. 9D includes plot 9D1 and illustrates induction of calcium signalingin SUM159 cells loaded with Fluo-4-AM before the sequential addition ofCCL5 (60 μg/mL) and fetal bovine serum (FBS) (5%), wherein RFIrepresents relative fluorescence intensities. The red traces are ofcells that did not respond to the addition of CCL5 ligand—but do respondto FBS (fetal bovine serum), the green line is the trace for the cellsthat do respond to the additionaof CCL5 , and also respond to FBS. Thisdata tells us that the SUM-159 cells are a population of cells in whichsome cells have the receptor and respond to CCL5− and some cells that donot have the CCR5 receptor and do not respond to its ligand CCL5;

FIG. 10A includes plots 10A1 and 10A2 and illustratesfluorescence-activated cell sorting (FACS) analysis for the abundance ofCCR5 receptor in either the SUM159-vector control cells or SUM159 cellsstably overexpressing the CCR5 receptor. APC-labeled antibody to CCR5was used to follow the CCR5 positive cells;

FIG. 10B illustrates schematic representation of the timeline forinduction of calcium signaling in which CCL5 is added to theSUM159-vector control cells or the SUM159 cells stably overexpressingthe CCR5 receptor at 60 seconds and FBS is added at 320 seconds;

FIG. 10C illustrates induction of calcium signaling (the Ca⁻² responseto CCL5 versus FBS) in the SUM159-vector control cells loaded withFluo-4-AM before and upon the addition of CCL5 or FBS;

FIG. 10D illustrates the average fluorescence for the population ofSUM159-vector control cells;

FIG. 10E illustrates induction of calcium signaling (the Ca⁻² responseto CCL5 versus FBS) in the SUM159 cells stably overexpressing the CCR5receptor at 60 seconds and FBS is added at 320 second;

FIG. 1OF illustrates the average fluorescence for the population ofSUM159-CCR5 cells;

FIG. 11A illurates that fact that maraviroc reduces MDA-MB-231 breastcancer lung metastasis burden as evidenced by the quantification resultsof weekly BLI, conducted for 5 weeks on maraviroc-treated NOD/SCID mice,wherein the lung metastatic tumor radiance antemortem was used as asurrogate measurement of tumor burden to the lungs (per FIG. 5B).

FIG. 11B illustrates the fact that maraviroc reduces MDA-MB-231 breastcancer lung metastasis burden as evidenced by the quantificationillustrates results of weekly BLI, conducted for 5 weeks onvehicle-treated NOD/SCID mice, wherein the lung metastatic tumorradiance antemortem was used as a surrogate measurement of tumor burden.

FIG. 12 includes plots 12A-12C and 12C1 and illustrates that fact thatpulmonary tumors are enriched in CCR5⁺⁴ cells as evidenced by the FACSanalysis of the proportion of CCR5+ cells into metastatic tumors causedby tail vein injection of MDA.pFULG cells. The proportion of CCR5+ cellswithin eGFP+MDA.pFULG cells in culture (FIG. 12A) was compared with thatof cells isolated from metastatic tumors (FIG. 12B). Analysis of data(FIG. 12C) showed an eight-fold increase in CCR5+ fraction in tumors(mean±SEM, n=6; Student's t test);

FIG. 13A includes plots 13A1 through 13A5 and illustrates cell surfaceexpression of CCR1 protein in cell lines MDA-MB-23, HS578T, SUM159,MCF-7, Jurkat cells by FACS analysis;

FIG. 13B includes plots 13B1 through 13B5 and illustrates cell surfaceexpression of CCR3 in cell lines MDA-MB-23, HS578T, SUM159, MCF-7,Jurkat cells by FACS analysis;

FIG. 13C includes plots 13C1 through 13C5 and illustrates cell surfaceexpression of CCR5 protein in cell lines MDA-MB-23, HS578T, SUM159,MCF-7, Jurkat cells by FACS analysis;

FIG. 13D shows relative abundance of cell surface expression of CCR1,CCR3 and CCR5 of cell lines MDA-MB-23, HS578T, SUM159, MCF-7, Jurkatcells as determined by FACS analysis;

FIG. 14A illustrates immunohistochemical staining of CCR5 in breastcancer tissue, showing staining was localized primarily to the breastcancer epithelial cell compared with normal breast tissue;

FIG. 14B illustrates immunohistochemical staining of CCR5 in normalbreast tissue, showing staining of CCR5 in normal breast tissue is verylow, demonstrating lack of CCR5 in normal breast compared with breasttumor;

FIG. 14C Immunohistochemical staining of CCR5 in breast cancer tissue,demonstrating CCR5 immunohistochemical staining being localizedprimarily to the breast cancer epithelial cell compared with normalbreast tissue.

FIG. 14D shows immunohistochemical staining of CCR5 in normal breasttissue, demonstrating lack of CCR5 in normal breast tissue (of differentpatients than shown in FIG. 14B) compared with breast tumor;

DETAILED DESCRIPTION OF THE INVENTION

Chemokine (C-C motif) ligand 5 (“CCL5”), also known as RANTES (anacronym for Regulated on Activation, Normal T cell Expressed andSecreted) is a protein which in humans is encoded by the CCL5 gene. Itsreceptor, C-C chemokine receptor type 5 (“CCR5” or “CD195”) is a proteinfound on the white blood cells. CCR5 is the main coreceptor used bymacrophage (M)-tropic strains of human immunodeficiency virus type 1(HIV-1) and HIV-2, which are responsible for viral transmission. CCR5therefore plays an essential role in HIV pathogenesis. A number ofinflammatory CC-chemokines, including MIP-1 alpha, MIP-1 beta, RANTES,MCP-2, and HCC-1[9-74] act as CCR5 agonists, while MCP-3 is a naturalantagonist of the receptor. CCR5 is mainly expressed in memory T-cells,macrophages, and immature dendritic cells, and is upregulated byproinflammatory cytokines.

Classes of antiretroviral medications with activity against HIV includenucleoside analogs, nonnucleoside reverse transcriptase inhibitors,protease inhibitors, fusion inhibitors, integrase inhibitors, and CCR5receptor antagonists. CCR5 antagonists exert their antiviral activityagainst HIV by blocking entry of CCR5 -tropic viruses into the CD4 Tcell. As a result, CCR5 antagonists have historically only beenassociated with expression in inflammatory cells in the immune system.

Prior to the present disclosure, the roles of the chemokine CCL5 and itsreceptor CCR5 in cancer progression were unclear. As disclosed herein,patients with cancers expressing CCL5 and its receptor CCR5, present newdrug targets. As disclosed herein, treating cancers that expressed CCL5and its receptor CCR5 with drugs that blocking their activitiesrespecting CCL5 and its receptor CCR5 selectively affect these cancercells that express mutant chemokine CCL5 and its receptor CCR5. In oneembodiment, a subpopulation of human breast cancer cell lines were foundto express CCR5 displayed a functional response to CCL5.

A microarray analysis conducted on 2,254 human breast cancer specimensfound increased expression of CCL5 and its receptor CCR5, but not CCR3,in the basal and HER-2 genetic subtypes. The subpopulation of humanbreast cancer cell lines found to express CCR5 displayed a functionalresponse to CCL5. Also, oncogene transformation induced CCR5 expression,and the subpopulation of cancer cells that expressed functional CCR5also displayed increased invasiveness.

In one embodiment, the CCR5 antagonists developed initially to blockCCR5 HIV co-receptor function, reduced in vitro invasion of cancer cellswithout affecting cell proliferation or viability. In one embodiment,the CCR5 antagonists include maraviroc and vicriviroc. In oneembodiment, the subpopulation of cancer cells that expressed functionalCCR5 include basal breast cancer cells. In one embodiment, the CCR5antagonists include maraviroc and vicriviroc reduced in vitro invasionof basal cancer cells without affecting the basal cancer cellproliferation or viability. In one embodiment, the CCR5 antagonistsinclude maraviroc and vicriviroc reduced in vivo invasion of basalcancer cells without affecting the basal cancer cell proliferation orviability.

However, as disclosed herein, CCL5 and its receptor CCR5 were found tobe expressed in cancer cells, including breast cancer cells, were alsofound to regulate cancer metastasis, spread of cancer from its primarysite to other sites in the body (e.g., brain, liver, lung). Moreover, itwas found that blocking the CCR5 receptor with CCR5 antagonists, such asMaraviroc and Vicriviroc, prevented migration and spread of the cancerfrom its primary site to other sites in the body.

As described herein, CCR5 and CCL5 were found to play a key role incancer invasiveness. For example, it was shown that CCR5 antagonistsslowed down and/or prevented the invasion of secondary sites in the bodyby cancers that express CCL5 and/or its receptor CCR5, demonstratingusefulness of CCR5 antagonists as viable adjuvant therapy for reducingthe risk of metastasis in cancer patients, including patients havingbasal breast cancer molecular subtype that express CCL5 and/or itsreceptor CCR5.

Cell receptor status can determine whether or not a cancer patient issusceptible to a particular anti-cancer treatment. For example, patientsdiagnosed with the basal breast cancer subtype, where the basal breastcancer subtype does not express the androgen or estrogen receptors orHER-2, current treatment choices, including chemotherapy, radiation, orsurgery, are not only the only choices for these patients, but all showpoor outcomes for these patients. Notwithstanding the fact thatcurrently there are no other effective therapies available to them, thepatients with this variant of basal breast cancer are furtherdisadvantaged by the fact that this variant of basal breast cancer isalso typically associated with metastasis. New cancer treatments presentthe only hope of cancer free survival for these patients. Accordingly,an urgent need exists for a specific targeted therapy for the basalbreast cancer subtype.

In one aspect the present invention relates to the use of CCR5modulators to treat, prevent, or manage a neoplasm or metastasis of theneoplasm. In one embodiment the neoplasm is cancer. Exemplary cancersand related disorders that can be treated, prevented, or managed inaccordance with the exemplary embodiments of the methods of the presentinvention include, but are not limited to, leukemias, such as but notlimited to, acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemias, such as, myeloblastic, promyelocytic, myelomonocytic,monocytic, and erythroleukemia leukemias and myelodysplastic syndrome;chronic leukemias, such as but not limited to, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cellleukemia; polycythemia vera; lymphomas such as but not limited toHodgkin's disease, non-Hodgkin's disease; multiple myelomas such as butnot limited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors such as but not limited to, glioma, astrocytoma, brain stemglioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas.

In one embodiment, antagonists of CCR5 are used to treat CCR5-expressingneoplasm or metastasis of the CCR5-expressing neoplasm. In oneembodiment, antagonists of CCR5 are used to prevent the neoplasm ormetastasis of said neoplasm. In one embodiment, antagonists of CCR5 areused to manage the neoplasm metastasis of said neoplasm. In oneembodiment, antagonists of CCR5 are used slow the progression of theCCR5-expressing neoplasm or metastasis of the CCR5-expressing neoplasm.In one embodiment, antagonists of CCR5 are used to delay metastasis ofthe CCR5-expressing neoplasm.

In one embodiment, antagonists of CCR5 that are suitable for use inaccordance with the exemplary methods of the present invention include,but are not limited to, the chemical compounds that are described inU.S. Pat. No. 6,667,314 by Perros et al. The chemical compounds ofPerros et al. and all formulations and dosage forms including them areincorporated by reference into the present application. Preferredexamples of the compounds by Perros et al. include:

-   -   4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide        (“Maraviroc”);    -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide;    -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide;    -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide;    -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;    -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarboxamide;        and pharmaceutically acceptable salts or solvates thereof.

In one embodiment, the modulators of CCR5 receptor that are suitable fortreating the neoplasm or metastasis of said neoplasm; or preventing theneoplasm or metastasis of said neoplasm; or managing the neoplasmmetastasis of said neoplasm; or slowing the progression of the neoplasmor metastasis of said neoplasm; or delaying the neoplasm or metastasisof said neoplasm; include, but are not limited to the chemical compoundsthat are described in U.S. Pat. No. 6,586,430 by Armour et al. Thechemical compounds of Armour et al. and all formulations or dosage formsincluding them are incorporated by reference into the presentapplication. Preferred examples of the compounds by Armour et al.include:

-   -   N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   N-{(1S)-3[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide;    -   1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidine        carboxamide;    -   1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3        .2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;    -   2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide;    -   2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo        [3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;    -   3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;    -   1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;    -   N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

-   -   1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;    -   1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;    -   N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   N-{(1S)-3[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex-3-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-1,2,4-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;    -   3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide;    -   N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;    -   N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;    -   2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;    -   N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;    -   N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;    -   2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;    -   N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;    -   N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;

-   -   1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   Methyl        3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;    -   N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl        }-1-propionyl-3-azetidinecarboxamide        1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;    -   2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;    -   1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;    -   1-Acetyl-N-{(1S)-3-[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;    -   N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;    -   N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;    -   2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;    -   1-Acetyl-N-{(1S)-3-[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N(1S)-3-[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   1-Acetyl-N-{(1S)-[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   (2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;    -   (2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;    -   2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;    -   N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;    -   2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   1-Acetyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   1-Methyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;    -   2-Methoxy-N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   N-{(1S)-3[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;        3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;    -   N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;    -   3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;    -   3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;    -   N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide;    -   1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

-   -   N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-methoxyacetamide;    -   N-{(1S)-3-[3        -exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;    -   N{-1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methoxypropanamide;    -   2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;        3-[Acetyl(methyl)amino]-N-{(1S)-3-[3exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;        and pharmaceutically acceptable salts thereof.

In yet another embodiment, modulators of CCR5 receptor that are suitablefor treating neoplasm or metastasis of the neoplasm; or preventing theneoplasm or metastasis of the neoplasm; or managing the neoplasmmetastasis of the neoplasm; or slowing the progression of the neoplasmor metastasis of the neoplasm; or delaying the neoplasm or metastasis ofsaid neoplasm; include, but are not limited to the chemical compoundsthat are described in U.S. Pat. Nos. 6,689,765 and 7,384,944 both byBaroudy et al. The chemical compounds of Baroudy et al. and allformulations or dosage forms including them are incorporated byreference into the present application. A preferred example of thecompounds by Baroudy et al. includes:(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanone(Vicriviroc, also previously named SCH 417690 and SCH-D), which has analternative IUPAC name of5-({4-[(3S)-4-{2-methoxy-1-[4-(trifluoromethyl)phenyl]ethyl}-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl}carbonyl)-4,6-dimethylpyrimidine;and pharmaceutically acceptable salts or solvates thereof.

In one aspect, the present invention provides a method of determiningwhether a subject has cancer or is at risk for developing cancer and/oris at risk for developing metastasis of In one embodiment, the methodincludes obtaining a biological sample from a subject having orsuspected of having cancer and assessing the level of expression of CCR5and/or of at least one of CCR5 ligands in the biological sample. In oneembodiment, the expression level of CCR5 and/or of at least one of CCR5ligands in the biological sample is compared to an expression level ofCCR5 and/or of at least one of CCR5 ligands in a control sample. In oneembodiment, if the expression level of CCR5 and/or of at least one ofCCR5 ligands in the biological sample is higher than the level ofexpression of CCR5 and/or of at least one of CCR5 ligands in the controlsample, then the subject is diagnosed as likely to have cancer. In oneembodiment, if the expression level of CCR5 and/or of at least one ofCCR5 ligands in the biological sample is higher than the level ofexpression of CCR5 and/or of at least one of CCR5 ligands in the controlsample, then the subject is diagnosed as at increased risk fordeveloping cancer. In one embodiment, if the expression level of CCR5and/or of at least one of CCR5 ligands in the biological sample ishigher than the level of expression of CCR5 and/or of at least one ofCCR5 ligands in the control sample, then the subject is diagnosed as atincreased risk for developing cancer metastasis.

The term “subject” as used herein is intended to include animals. Inparticular embodiments, the subject is a mammal, a human or nonhumanprimate, a dog, a cat, a horse, a cow or a rodent.

In another aspect, the present invention provides a method for molecularclassification of cancer based on a level of expression of CCR5 and/orof at least one of CCR5 ligands in the biological sample of the cancer.In one embodiment, the method for molecular classification of cancercomprises (a) obtaining a biological sample of cancer from subject; (b)determining level of expression of CCR5 and/or level of expression of atleast one of CCR5 ligands in the biological sample; and (c) if the levelof expression of CCR5 and/or level of expression of at least one of

CCR5 ligands determined in step (b) is higher than the level ofexpression of CCR5 and/or of at least one of CCR5 ligands in a controlsample, then the cancer is classified as CCR5 -expressing cancer. In oneembodiment, a cancer subject whose cancer has been classified as CCR5-expressing cancer is diagnosed as likely at risk for developingmetastasis of the cancer. In one embodiment, the cancer is breastcancer. In one embodiment, the cancer is prostate cancer. In oneembodiment, the cancer is selected from the group consisting ofleukemias, such as but not limited to, acute leukemia, acute lymphocyticleukemia, acute myelocytic leukemias, such as, myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemiasand myelodysplastic syndrome; chronic leukemias, such as but not limitedto, chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor; esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to pappillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas.

In another aspect, the present invention provides for a method oftreating, preventing, or managing a CCR-5 expressing neoplasm or ametastasis of the CCR5-expressing neoplasm in a subject. In oneembodiment, the a method of treating or managing a CCR-5 expressingneoplasm or a metastasis of the CCR5-expressing neoplasm in subjecthaving the CCR5-expressing neoplasm or at risk for developing metastasisof the CCR5-expressing neoplasm, comprises administering to the subjecta CCR5 modulator. In one embodiment, the CCR5 modulator comprises a CCR5antagonist. In one embodiment, the CCR5 antagonist comprises4,4-difluoro-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide(“Maraviroc”). In one embodiment, the CCR5 modulator comprises a CCR5antagonist. In one embodiment, the CCR5 antagonist comprises(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanone(“Vicriviroc”). In one embodiment, the CCR5 antagonist antagonist isselected from the group consisting of4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide(“Maraviroc”) and(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanone(“Vicriviroc”).

In one embodiment, sutiable CCR5 antagonist is selected from the groupconsisting of: 4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide(“Maraviroc”);

-   -   N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide;

N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide;

N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide;

N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;

N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarboxamide;and pharmaceutically acceptable salts or solvates thereof.

In one embodiment, suitable CCR5 antagonist is selected from the groupconsisting of:

N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;

N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;

N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide;

1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;

1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide;

2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;

3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;

1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;

1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;

1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

N-{(1S)-3[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex-3-yl]-1-phenylpropyl}cyclobutanecarboxamide;

2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-1,2,4-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;

2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;

3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide;

N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;

2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;

N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;

2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;

N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;

N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;

2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;

N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;

N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;

1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

Methyl3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;

N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide1-Acetyl-N-{(1S-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;

2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;

1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;

1-Acetyl-N-{(1S)-3[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;

N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;

N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;

2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;

1-Acetyl-N-{(1S)-3[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N(1S)-3[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-propionyl-3-azetidinecarboxamide;

1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

1-Acetyl-N-{(1S)-3[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;

N-{(1S)-3[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

(2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;

(2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;

2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;

N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;

2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

1-Methyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

2-Methoxy-N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

N-{(1S)-3[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;

N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;

3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;

N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;

3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide;

1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-methoxyacetamide;

N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

N{-1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methoxypropanamide;

2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;

3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;and pharmaceutically acceptable salts thereof.

As disclosed herein, one aspect of the present invention provides for amethod of treating, preventing, or managing a CCR-5 expressing neoplasmor a metastasis of the CCR5-expressing neoplasm in a subject byadministering to the subject a CCR5 receptor antagonist. In oneembodiment, CCR5 receptor anatagonists are administered in the form ofpharmaceutical formulations or dosage forms that include the CCR5receptor antagonists. In one embodiment, the CCR5 receptor antagonistsare used in the form of acids, esters, or other suitable chemicalderivatives. In one embodiment, the CCR5 receptor antagonists are in theform of pharmaceutically acceptable salts derived from various organicand inorganic acids and bases in accordance with procedures well knownin the art. In one embodiment, the expression “pharmaceuticallyacceptable salt” as used herein is intended to mean an active ingredientcomprising a CCR5 receptor antagonist utilized in the form of a saltthereof, especially where the salt form confers on the CCR5 receptorantagonist improved pharmacokinetic properties as compared to the freeform of the CCR5 receptor antagonist or other previously disclosed saltform. In one embodiment, a pharmaceutically acceptable salt form of theCCR5 receptor antagonist may also initially confer a desirablepharmacokinetic property on the CCR5 receptor antagonist which it didnot previously possess, and may even positively affect thepharmacodynamics of the CCR5 receptor antagonist with respect to itstherapeutic activity in the body. In one embodiment, the pharmacokineticproperties of the CCR5 receptor antagonist which may be favorablyaffected include, e.g., the manner in which the CCR5 receptor antagonistis transported across cell membranes, which in turn may directly andpositively affect the absorption, distribution, biotransformation orexcretion of the CCR5 receptor antagonist.

While the route of administration of the pharmaceutical composition isimportant and various anatomical, physiological and pathological factorscan critically affect bioavailability, the solubility of the CCR5receptor antagonist is usually dependent upon the character of theparticular salt form thereof which it utilized. Further, an aqueoussolution may provide the most rapid absorption of an active ingredientinto the body of a patient being treated, while lipid solutions andsuspensions, as well as solid dosage forms, may result in less rapidabsorption. Oral ingestion of the CCR5 receptor antagonist is the mostpreferred route of administration for reasons of safety, convenience,and economy, but absorption of such an oral dosage form can be adverselyaffected by physical characteristics such as polarity, emesis caused byirritation of the gastrointestinal mucosa, destruction by digestiveenzymes and low pH, irregular absorption or propulsion in the presenceof food or other drugs, and metabolism by enzymes of the mucosa, theintestinal flora, or the liver. Formulation of the CCR5 receptorantagonist into different pharmaceutically acceptable salt forms may beeffective in overcoming or alleviating one or more of the above-recitedproblems encountered with absorption of oral dosage forms. Well-knownpharmaceutically acceptable salts include, but are not limited toacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,besylate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecysulfate, ethanesulfonate,fumarate, glucoheptanoate, gluconate, glycerophosphate, hemisuccinate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, isethionate,lactate, lactobionate, maleate, mandelate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate,pectinate, persulfate, 3-phenylpropionate, phosphonate, picrate,pivalate, propionate, salicylate, sodium phosphate, stearate, succinate,sulfate, sulfosalicylate, tartrate, thiocyanate, thiomalate, tosylate,and undecanoate.

Base salts of the compounds of suitable CCR5 receptor antagonistsinclude, but are not limited to ammonium salts; alkali metal salts suchas sodium and potassium; alkaline earth metal salts such as calcium andmagnesium; salts with organic bases such as dicyclohexylamine,meglumine, N-methyl-D-glucamine, trishydroxymethyl)methylamine(tromethamine), and salts with amino acids such as arginine, lysine,etc. Compounds of the present invention which comprise basicnitrogen-containing groups may be quaternized with such agents as (C₁C₄)alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides,bromides and iodides; di(C₁-C₄) alkyl sulfate, e.g., dimethyl, diethyland diamyl sulfates; (C₁₀-C₁₈) alkyl halides, e.g., decyl, dodecyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaryl-(C₁-C₄) alkyl halides, e.g., benzyl chloride and phenethyl bromide.Such salts permit the preparation of both water-soluble and oil-solublecompounds of the present invention.

Among the above-recited pharmaceutical salts those which are preferredinclude, but are not limited to acetate, besylate, citrate, fumarate,gluconate, hemisuccinate, hippurate, hydrochloride, hydrobromide,isethionate, mandelate, meglumine, nitrate, oleate, phosphonate,pivalate, sodium phosphate, stearate, sulfate, sulfosalicylate,tartrate, thiomalate, tosylate, and tromethamine.

Multiple salts forms are included within the scope of the presentinvention where a CCR5 receptor antagonist ontains more than one groupcapable of forming such pharmaceutically acceptable salts. Examples oftypical multiple salt forms include, but are not limited to bitartrate,diacetate, difumarate, dimeglumine, diphosphate, disodium, andtrihydrochloride. Suitable CCR5 receptor antagonists can be administeredalone but will generally be administered in admixture with one or moresuitable pharmaceutical excipients, diluents or carriers selected withregard to the intended route of administration and standardpharmaceutical practice.

For example, a CCR5 receptor antagonist can be administered orally orsublingually in the form of tablets, capsules, ovules, elixirs,solutions or suspensions, which may contain flavouring or colouringagents, for immediate or controlled release applications. Such tabletsmay contain excipients such as microcrystalline cellulose, lactose,sodium citrate, calcium carbonate, dicalcium phosphate and glycine,disintegrants such as starch (preferably corn, potato or tapiocastarch), alginic acid and certain complex silicates, and granulationbinders such as polyvinylpyrrolidone, sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, sodiumlauryl sulfate and talc may be included. Solid compositions of a similartype may also be employed as fillers in gelatin capsules. Preferredexcipients in this regard include lactose or milk sugar as well as highmolecular weight polyethylene glycols. For aqueous suspensions and/orelixirs, the CCRS receptor antagonist may be combined with varioussweetening or flavouring agents, colouring matter or dyes, withemulsifying and/or suspending agents and with diluents such as water,ethanol, propylene glycol and glycerin, and combinations thereof

Suitable CCR5 receptor antagonists can also be injected parenterally,for example, intravenously, intraperitoneally, intrathecally,intraventricularly, intrastemally, intracranially, intramuscularly orsubcutaneously, or they may be administered by infusion techniques. Theyare best used in the form of a sterile aqueous solution which maycontain other substances, for example, enough salts or glucose to makethe solution isotonic with blood. The aqueous solutions should besuitably buffered (preferably to a pH of from 3 to 9), if necessary. Thepreparation of suitable parenteral formulations under sterile conditionsis readily accomplished by standard pharmaceutical techniques well knownto those skilled in the art.

For oral and parenteral administration to human subjects, the dailydosage level of the CCR5 receptor antagonist will usually be from 1microgram/kg to 25 mg/kg (in single or divided doses). Thus tablets orcapsules of the CCR5 receptor antagonist may contain from 0.05 mg to 1.0g of active compound for administration singly or two or more at a time,as appropriate. The physician in any event will determine the actualdosage which will be most suitable for any individual patient and itwill vary with the age, weight and response of the particular patient.The above dosages are exemplary of the average case. There can, ofcourse, be individual instances where higher or lower dosage ranges aremerited and such are within the scope of this invention. Suitable CCR5receptor antagonists can also be administered intranasally or byinhalation and are conveniently delivered in the form of a dry powderinhaler or an aerosol spray presentation from a pressurised container ora nebuliser with the use of a suitable propellant, egdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, a hydrofluoroalkane such as1,1,1,2-tetrafluorethane (HFA 134a), carbon dioxide or other suitablegas. In the case of a pressurised aerosol, the dosage unit may bedetermined by providing a valve to deliver a metered amount. Thepressurized container or nebulizer may contain a solution or suspensionof the active compound, eg using a mixture of ethanol and the propellantas the solvent, which may additional contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a CCR5 receptor antagonist and a suitable powder base such aslactose or starch.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or “puff” contains from 20 μg to 20 mg of a CCR5 receptorantagonist for delivery to the subject. The overall daily dose with anaerosol will be in the range of from 20 μg to 20 mg which may beadministered in a single dose or, more usually, in divided dosesthroughout the day. Alternatively, Suitable CCR5 receptor antagonistscan be administered in the form of a suppository or pessary, or they maybe applied topically in the form of a lotion, solution, cream, ointmentor dusting powder. The CCR5 receptor antagonist may also betransdermally administered by the use of a skin patch. They may also beadministered by the ocular route, particularly for treating neurologicaldisorders of the eye.

For ophthalmic use, the compounds can be formulated as micronisedsuspensions in isotonic, pH adjusted, sterile saline, or, preferably, assolutions in isotonic, pH adjusted, sterile saline, optionally incombination with a preservative such as benzylalkonium chloride.Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the compounds of the formula (I)can be formulated as a suitable ointment containing the active compoundsuspended or dissolved in, for example, a mixture with one or more ofthe following: mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, they can be formulated as a suitablelotion or cream, suspended or dissolved in, for example, a mixture ofone or more of the following: mineral oil, sorbitan monostearate, apolyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax,cetearyl alcohol, 2-octyldodecanol, benyl alcohol and water.

In some embodiments, the compounds described herein can modulate CCR5chemokine receptor activity and consequent or associated pathogenicprocesses subsequently mediated by the CCR5 receptor and its ligands.The expression “modulate CCR5 chemokine receptor activity” as usedherein is intended to refer to manipulation of the basic physiologicalprocesses and agencies which involve CCR5 chemokine receptors and theirligands. Included within the scope of this intended meaning are alltypes and subtypes of CCR5 receptors, in whatever tissues of aparticular patient they are found, and in or on whatever components ofthe cells comprising those tissues they may be located. Most commonly,CCR5 receptors are situated on the cell membranes of particular celltypes such as monocytes. CCR5 receptors participate in and define, alongwith various endogenous ligands to which they are naturally bound,signaling pathways which control important cellular and tissue functionsby means of the influence which they exert on the movement of agentssuch as the chemokines, into and out of those cells and tissues.

The dosage and dose rate of the compounds of Formula (I) effective fortreating or preventing diseases and conditions in a patient which aremediated by or associated with modulation of CCR5 chemokine receptoractivity as described herein, as well as for favorably affecting theoutcome thereof in the patient, in accordance with the methods oftreatment of the present invention comprising administering to thepatient a therapeutically effective amount of a CCR5 receptorantagonist, will depend on a variety of factors such as the nature ofthe CCR5 receptor antagonist, the size of the patient, the goal of thetreatment, the nature of the pathology being treated, the specificpharmaceutical composition used, the concurrent treatments that thepatient may be subject to, and the observations and conclusions of thetreating physician.

Generally, however, the effective therapeutic dose of a suitable CCR5receptor antagonist which will be administered to a subject will bebetween about 10 μg (0.01 mg)/kg and about 60.0 mg/kg of body weight perday, preferably between about 100 μg (0.1 mg)/kg and about 10 mg/kg ofbody weight per day, more preferably between about 1.0 mg/kg and about6.0 mg/kg of body weight per day, and most preferably between about 2.0mg/kg and about 4.0 mg/kg of body weight per day of the CCR5 receptorantagonist.

Included within the scope of the present invention are embodimentscomprising coadministration of, and compositions which contain, inaddition to a CCR5 receptor antagonist as active ingredient, additionaltherapeutic agents and active ingredients. Such multiple drug regimens,often referred to as combination therapy, may be used in the treatmentand prevention of any of the diseases or conditions mediated by orassociated with CCR5 chemokine receptor modulation, particularly cancermetastasis. The use of such combinations of therapeutic agents isespecially pertinent with respect to the treatment, prevention, ormanagement of cancer metastasis within a subject in need of treatmentcancer, prevention cancer, or management of risk of cancer metastasis.

Exemplary CCR5 receptor antagonists may be administered in accordancewith a regimen of 1 to 4 times per day, preferably once or twice perday. The specific dose level and frequency of dosage for any particularpatient may be varied and will depend upon a variety of factorsincluding the activity of the specific compound employed, the metabolicstability and length of action of that compound, the age, body weight,general health, sex, diet, mode and time of administration, rate ofexcretion, drug combination, the severity of the particular condition,and the host undergoing.

As disclosed in U.S. Pat. No. 6,667,314 by Perros et al. CCR5antagonists can be administered orally, buccally or sublingually in theform of tablets, capsules, multi-particulates, gels, films, ovules,elixirs, solutions or suspensions, which may contain flavouring orcolouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications. The CCR5 receptorantagonists may also be administered as fast-dispersing orfast-dissolving dosage forms or in the form of a high energy dispersionor as coated particles. Suitable formulations of the compounds of theCCR5 receptor antagonist may be in coated or uncoated form, as desired.Such solid pharmaceutical compositions, for example, tablets, maycontain excipients such as microcrystalline cellulose, lactose, sodiumcitrate, calcium carbonate, dibasic calcium phosphate, glycine andstarch (preferably corn, potato or tapioca starch), disintegrants suchas sodium starch glycollate, croscarmellose sodium and certain complexsilicates, and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),sucrose, gelatin and acacia. Additionally, lubricating agents such asmagnesium stearate, stearic acid, glyceryl behenate and talc may beincluded.

“Metastasis”, as used herein, is defined as the transfer of malignanttumor cells, or neoplasm, via the circulatory or lymphatic systems orvia natural body cavities, usually from the primary site of neoplasia toa distant site in the body, and subsequent development of secondarytumors or colonies in the new location. In some exemplary embodiments ofthe methods of the present invention, metastasis comprises a tumormetastasis in or more organs selected from the group consisting ofliver, brain, bladder, lung, adrenal gland, kidney, bone, skin orpancreas or control kidney and combinations thereof.

In another aspect, the present invention provides a method ofidentifying a compound that reduces or prevents or treats cancermetastasis. In one embodiment, the method identifies a candidatecompound that selectively interferes with proliferation or viability ofneoplastic cells that over express CCR5 and/or over express at least oneof CCR5 receptor ligand. In one embodiment, the method identifies acandidate compound that selectively blocks activity of CCR5 and/or atleast one of CCR5 receptor ligand in neoplastic cells that over expressCCR5 and/or over express at least one of CCR5 receptor ligand. In oneexemplary embodiment, the method for identifying a candidate compoundthat reduces or prevents or treats cancer metastasis in neoplastic cellsthat over express CCR5 and/or over express at least one of CCR5 receptorligand, comprises (a) contacting a one or more neoplastic cells thatover express CCR5 and/or over express at least one of CCR5 receptorligand with one or more candidate compounds; and (b) detecting activityof and/or proliferation or viability of the one or more neoplastic cellsthat over express CCR5 and/or over express at least one of CCR5 receptorligand, wherein decreased activity and/or decreased proliferation and/ordecreased viability of the one or more neoplastic cells (relative to ascompared to a control sample) identifies the candidate compound as acompound that that selectively reduces or prevents or treats cancermetastasis in neoplastic cells that over express CCR5 and/or overexpress at least one of CCR5 receptor ligand. In one embodiment, ifproliferation of the one or more neoplastic cells is decreased/depressedcompared to untreated (control) neoplastic cells that over express CCR5and/or over express at least one of CCR5 receptor ligand, the candidatecompound is identified as a compound that selectively reduces orprevents or treats metastasis of a neoplasm cells that over express CCR5and/or over express at least one of CCR5 receptor ligand. In oneembodiment, if viability of the one or more neoplastic cells isdecreased/depressed compared to untreated (control) neoplastic cellsthat over express CCR5 and/or over express at least one of CCR5 receptorligand, the candidate compound is identified as a compound that thatselectively reduces or prevents or treats metastasis of a neoplasm cellsthat over express CCR5 and/or over express at least one of CCR5 receptorligand. In one embodiment, the one or more CCR5 receptor ligandscomprise CCLS. In one embodiment, the one or more CCR5 receptor ligandscomprise CCLS. In one embodiment, the one or more CCR5 receptor ligandscomprise CCL7.

For the study disclosed herein, CCL5 and CCR5 expression in human breastcancer cell lines were investigated as well as the effect of CCR5antagonists in vitro and in vivo. An interrogation was conducted using amicroarray dataset to evaluate CCR5 and CCL5 expression in the contextof 2,254 patient breast cancer samples. Samples in the dataset wereassigned to five breast cancer subtypes, including luminal A, luminal B,normal-like, basal and HER-2 overexpressing disease. The analysisrevealed an increased expression of CCL5 and CCR5 in patients with basaland HER-2 subtypes. 58% the cancer samples indicated a positive CCR5 andCCL5 signature. It was found that oncogenes turn on the CCR5 receptor innormal breast cells as they became transformed into cancer cells.Metastasis those cells was also found to be regulated by CCR5.

To evaluate the functional relevance of CCR5 in cellular migration andinvasion in vitro, the drugs were tested in 3-D invasion assays with twodifferent cell lines. It was found that both antagonists inhibitedbreast cancer cell invasiveness.

To evaluate the functional relevance of CCR5 in cellular migration andinvasion in vivo, mice were injected with the antagonists andinvasiveness of the basal breast cancer cells to other tissue, i.e.lung, was tracked with bioluminescence imaging. It was found that micetreated with the drug showed a more than 90% reduction in both thenumber and size of pulmonary metastases compared to untreated mice. Thisand the other preclinical studies provide the rational basis forstudying the use of CCR5 antagonists as new treatments to block thedissemination of basal breast cancers. These findings may also haveimplications for other cancers where CCR5 promotes metastasis, such asprostate and gastric.

Referring to the drawings in detail, wherein like reference numeralsindicate like elements throughout, there are exemplary embodiments ofthe present invention shown in FIGS. 1A-14D.

Referring to FIG. 1A, there is shown a heatmap of the expression of CCL5and its receptor CCR5 in samples from patients with breast cancerdivided by molecular subtypes of breast cancer, namely luminal A,luminal B, basal, normal-like, and Her-2, based on their gene expressionpattern. The heatmap shows that relative abundances of CCL5 and CCR5 areincreased (over expressed) in patients with the basal and HER-2 breastcancer subtypes. The heatmap also shows that CCL5 and CCR5 are overexpressed in the Her-2 breast cancer subtype.

Referring to FIG. 1B, there is shown fluorescence-activated cell sorting(FACS) scatter plots and correlation analysis (Student t test) of theexpression of CCL5 and CCR5 among the breast cancer molecular subtypeswhose expression of CCL5 and its receptor CCR5 is shown FIG. 1A.Consistent with the expression of CCL5 and its receptor CCR5 observed inA, the scatter plots show CCL5 and CCR5 are over expressed in patientswith the basal and HER-2 breast cancer subtypes.

Referring to FIG. 1C, there is shown quantification of the proportionsof the breast cancer samples overexpressing CCL5 and CCR5 (fraction ofthe bars representing upper right quadrants of the scatter plots shownin Figure B) displayed in FIG. 1B. The number of samples in each subtypeis indicated at the top of the bar.

Referring to FIG. 1D, there is shown metastasis-free Kaplan-Meier plotsand log-rank analysis for the different genetic subtypes in the analyzeddatabase described in Materials and Methods section of the presentdisclosure. The metastasis-free Kaplan-Meier plots show that patientswith the basal or HER-2 subtypes of breast cancer display increasedprobability to develop metastasis.

FIGS. 2A-2H, show that human breast cancer cell lines that express CCR5respond to CCL5. In FIG. 1A, flow cytometric histograms of the CCR5expression in MDA-MB-231 breast cancer cells identified a subpopulationof CCR5+ cells. In FIG. 2B, induction of calcium signaling in cellsloaded with Fluo-4-AM before the sequential addition of CCL5 (60 μg/mL)and FBS (5%) is shown. A fraction of cells responded to CCL5 (closedarrowheads in the middle of micrographs) whereas the rest did not (openarrowheads). The average changes in fluorescence on 5 responsive (greenline) and 5 nonresponsive (red line) cells are represented in the farright graphs. Data shown are representative of 3 to 5 independentexperiments for each cell line (Bar, 100 μm). In FIG. 2C, 3D invasioninto collagen gels by breast cancer cell lines, using CCL5 (15 μg/mL) aschemoattractant is shown. Figure D shows mean distances of invasion±SEMfrom 3 independent experiments whose 3D invasion are shown in FIG. 2C.FIG. 2E shows 3D invasion assays, and their corresponding quantificationshown in FIG. 2F (mean±SEM, n=3), for MCF-10A cells and MCF-10A-NeuT,-Ras, and -Src derivatives showing that CCL5 -induced invasion isactivated by oncogenic transformation. FIG. 2G shows CCR5+ cells displayincreased invasiveness. CCR5+ and CCR5− subpopulations from SUM-159 cellline were isolated by FACS and invasion into collagen gels was evaluatedusing FBS as chemoattractant. Quantification of FACS and invasion intocollagen gels experiments of the samples in FIG. 2G is shown in FIG. 2Has mean±SEM of 2 independent experiments. Statistical analysis wasconducted using the Student t test.

FIGS. 3A-3D show that CCR5 antagonists block CCL5 -induced calciumsignaling. In FIG. 3A, there is shown intensity versus time analysis ofFluo-4 AM-loaded MDA-MB-231 cells treated with the CCR5 antagonistsmaraviroc or vicriviroc (100 nmol/L) for 30 minutes before the additionof CCL5 (60 μg/mL). Micrographs illustrate the axis (x-x′) of thepseudoline scan plot. Those axes were used to construct the adjacentintensity versus time plots. In FIG. 3B, there is shown comparison ofthe fraction of cells with increased fluorescence intensity uponaddition of

CCL5. In FIG. 3C, CCL5 -induced calcium signaling was also blocked byCCR5 antagonists in Hs578T cells. The corresponding quantification isshown in FIG. 3D. The data in FIGS. 3B and 3D are mean±SEM of 3 to 4independent experiments. Statistical analysis was conducted using theStudent t test.

FIGS. 4A-4D show that CCR5 antagonists block FBS-induced breast cancercell invasion. In FIG. 4A, there is shown 3D reconstruction ofFBS-induced invasion into collagen gels by Hs578T breast cancer cells inpresence of CCR5 antagonists (100 nmol/L). In FIG. 3C there is shown 3Dreconstruction of FBS-induced invasion into collagen gels by SUM-159breast cancer cells in presence of CCR5 antagonists (100 nmol/L). Thecorresponding quantifications (mean±SEM, n=3) and analysis (Bonferroni ttest) are displayed in FIGS. 4B and 4D.

Referring to FIGS. 5A-5F, the data presented show that the CCR5antagonist maraviroc inhibits lung metastases in vivo. In FIG. 5A,MDA-MB-231 cells transduced with Luc2-eGFP fusion protein were injectedinto the tail vein of NOD/SCID mice and the in vivo bioluminescentsignal was quantified weekly. Representative in vivo images of vehicle-or maraviroc-treated (8 mg/kg every 12 hours) mice are shown in FIG. 5A.In FIG. 5B, there is shown quantification (mean±SEM, n=6) of BLI in thecontrol (red line) and treated groups (blue line. Statistical comparison(*, P =0.048) was carried out using Student t test with Welch correctionfor heterogeneous variances. As shown in FIG. 5C, the presence ofpulmonary tumors and the differences between treatments werecorroborated by ex vivo imaging (left) and India ink staining (right).As shown in FIG. 5D, the fraction of mice with metastatic tumors wassignificantly larger in the control group (P<0.0001, Fisher exact test).FIG. 5E shows histologic analysis (hematoxylin and eosin staining, ×100)and the corresponding quantification (shown in FIG. 5F) of the areacovered by metastatic tumors in lung slides. Tumor area was quantifiedwith the Nikon Elements BR 3.0 software analyzing at low magnification(×40) 2 random fields of 2 different histologic sections (separated 600μm from each other) per mouse. Statistical analysis was conducted usingthe Student t test with Welch correction for heterogeneous variances(n=33 and 12 for control and treated groups, respectively).

Referring to FIGS. 6A-6H, it is shown that maraviroc reduces lungcolonization but does not modify cell proliferation. In FIG. 6A, thereis shown the effect of CCR5 antagonist on breast cancer cell viability.MDA-MB-231 cells were exposed to increasing concentrations of maraviroc(inverted triangles) or vicriviroc (squares) for 48 hours and the cellviability was evaluated by MTT assay. Graph is from a representativeexperiment carried out by sextuplicate. No statistical differences werefound (ANOVA) in 3 independent experiments. In FIG. 6B, there is shownCCR5 expression in MDA-MB-231 cells stably transfected withpcDNA3.1+/Zeo+ (MDA.Vector) or human CCR5 cloned into pCDNA3+Zeo+(MDA.CCRS). In FIG. 6C, comparison of in vitro proliferation rates ofMDA.Vector versus MDA.CCR5 showed no differences (ANOVA). Representativeexperiment from 2 carried out by sextuplicate. In FIG. 6D, to evaluatethe in vivo effect of maraviroc on growth of established metastasis,treatment of mice was initiated 10 days after injection of MDA.pFULGcells as illustrated. In FIG. 6E, quantification (mean ±SEM, n=5) of invivo BLI in the control (red/squares) and treated groups(blue/triangles) showed no differences in the growth rate. There is noeffect of maraviroc of the growth rate of the tumors once established inthe lungs. Thus, the reduction in tumor mass in the lungs is due to theinhibition of “homing” or spread of tumors to the lung. FIG. 6F showsschema of the experimental design used to evaluate CCR5 role in lungcolonization. FIG. 6G shows representative confocal images andquantification (FIG. 6H) of the number of eGFP+ cells in lungs 24 hoursafter injection of MDA.pFULG cells. Cells expressing eGFP were countedin 3 random fields of 2 different histologic sections (separated 700 μmfrom each other) per mouse (n=5 mice per group). Statistical analysiswas conducted using Student t test. Bar in micrographs, 100 μm.

Reference is now made to FIGS. 7A-7D. To determine the gene expressionsignaling pathway associated with enrichment of CCR5 and CCL5, GSEAanalysis using KEGG and GO was conducted of the tumor samples discussedherein. FIG. 7A shows the GSEA analysis. These studies showed enrichmentfor gene expression of pathways including lymphocyte activation,Janus-activated kinase (JAK)-STAT signaling, and Toll-like receptoractivation. The receptors for CCL5 include CCR1 and CCR3. Increasedexpression of CCL5 associated with increased CCR1, but not CCR3, in thebasal and HER-2 genetic tumor type (FIGS. 7B and 7C). In ER-negativepatients receiving chemotherapy, there was an insignificant trend towardreduced metastasis-free survival and relapse-free survival in theincreased CCR5 population, compared with the population with reducedCCR5 expression (FIG. 7D).

In FIGS. 8A-8D, a comparison of expression levels for CCL5 versus CCR5,CCR1, and CCR3, comparing normal breast with breast cancer showedincreased correlation between receptor and ligand expression levels intumors compared with healthy breast tissue. FIG. 8A illustrates heatmapof CCL5 expression and its receptors CCR5, CCR1 and CCR3 in healthypopulation. The healthy population was previously described as part of acollection of 2550 breast cancers (Ertel, A., Dean, J. L., Rui, H., Liu,C., Witkiewicz, A. K., Knudsen, K. E., and Knudsen, E. S. RB-pathwaydisruption in breast cancer: differential association with diseasesubtypes, disease-specific prognosis and therapeutic response. CellCycle, 9: 4153-4163, 2010.).

Referring to FIGS. 9A-9D, three human breast cancer cell lines with abasal phenotype and molecular signature: MDA-MB-231, Hs578T, and SUM-159(34-37) were used as models in the studies. Analysis of CCR5 expressionby FACS showed that a small subpopulation of cells were positive for thereceptor in all 3 cell lines (FIG. 2A for MDA-MB-231 and FIG. 9A andFIG. 9C for Hs578T and SUM-159). Because CCR5 activation induces calciumflux (38, 39), the activation of calcium signaling was assessed by CCL5.Addition of CCL5 to the cultures induced immediate calcium fluxes in asubpopulation of cells (FIG. 2B for MDA-MB-231 and FIGS. 9B and 9D forHs578T and SUM-159), providing evidence that CCR5 is functional in basalbreast cancer cells. As a positive control, the same cultures wereexposed to 5% FBS (40). Calcium flux, assessed by relative fluorescenceintensity, increased in more than 95% of the cells after FBS addition(FIG. 2B and FIGS. 9B and 9D). To further distinguish CCL5 -dependentsignaling, SUM159 cells were stably transduced with a CCR5 expressionvector and the Ca⁺² response to CCL5 versus FBS was conducted (FIGS. 10C and 10D vs. FIGS. 10E and 10F). CCR5 induced Ca⁺² signaling in theCCR5 -overexpressing cells, whereas both lines responded similarly toFBS induced Ca⁺² activation (FIGS. 10A-10F).

In view of the finding that CCR5 inhibition by CCR5 antagonists reducedcalcium signaling and cell invasion, the in vivo effect of maraviroc onlung metastasis was determined. MDA-MB-231 cells were transduced withinthe Luc2-eGFP lentiviral vector (MDA.pFULG cells) in an experimentalmetastasis model. The Luc2 gene is a codon-optimized version of Luc andcells expressing this reporter were 10 to 100 times brighter than theunmodified Luc gene (30). After injection of MDA.pFULG cells into thetail vein of mice, noninvasive BLI enabled the early detection of breastcancer metastasis (41). Weekly BLI was conducted for 5 weeks and theradiance antemortem was used as a surrogate measurement of tumor burden.Mice treated with maraviroc (8 mg/kg twice daily) showed a significantreduction in both the number and the size of pulmonary metastasescompared with vehicle-treated mice (FIGS. 5A and 5B, FIGS. 11A and 11B).To avoid the possibility that metastases were missed because ofinappropriate imaging, ex vivo imaging, India ink staining (FIG. 5C),and histology (FIG. 5E) of the lungs were conducted. Histologic analysiscorroborated that tumor burden corresponds to bioluminescence, aspreviously shown (30). Metastatic tumors were still detectable in 50% ofthe maraviroc-treated mice, but their mean size was reduced by 65%(FIGS. 5D and 5F). Interestingly, analysis of CCR5 expression in lungsfrom control mice showed an 8-fold enrichment of the CCRS+fraction (FIG.12). Collectively, these results provide evidence that CCR5 antagonistsreduce breast cancer metastasis in vivo.

Given the aggressive clinical behavior of basal breast cancer and thelack of targeted therapies for it, the importance of the CCLS/CCR5 wasevaluated in invasion and metastasis in the human breast cancer celllines MDA-MB-231, Hs578T and SUM-159. These cell lines reflect theclinicopathologic features of the basal subtype of breast cancer(including the lack of HER-2, ER, and progesterone receptor), abasal-like molecular signature, the activation of specific signalingpathways (e.g., hypoxic or EGF receptor responses) and over expressionof epithelial-mesenchymal transition proteins (FN, VIM, ad matrixmetalloproteinase 2; refs. 34-37). Only a small fraction of cells withinthe cell lines used in this study expressed CCR5 as evaluated by FACSanalysis. The studies confirmed the expression of CCR5 in MDA-MB-23cells by reverse transcriptase PCR and showed the presence of the CCR5protein by FACS analysis (FIGS. 13A-13D), and showed that CCR5immunohistochemical staining was localized primarily to the breastcancer epithelial cell, compared with normal breast tissue (FIGS.14A-14D).

The results disclosed herein show that CCL5 activates calcium flux inbasal-like human breast cancer cells. By using the selectiveCCR5-antagonists maraviroc and vicriviroc (both with IC50 below 30nmol/L; refs. 44, 45), it was shown that CCLS-activated signaling ismediated by CCRS. However, the fraction of CCL5-responsive cells (10%and 12% for MDA-MB-231 and Hs578T cells, respectively) is higher thanthe percentage of CCR5-expressing cells determined by FACS. This may bedue to the greater sensitivity of the Ca2+ activation assays comparedwith the sensitivity of analysis by FACS. In addition, CCL5 -inducedcalcium redistribution is not completely blocked by CCR5 antagonists.This may be caused by the expression of other receptors to CCL5, namelyCCR1 and CCR3. CCR5 has been identified as the main CCL5 receptor inMDA-MB-231 cells (13) and CCR1 and CCR3 transcripts are absent in bothMDA-MB-231 or Hs578T cell lines (8) and breast tumor samples (11). CCR1and CCR3 were able to be detected by FACS (FIGS. 13A-13D), suggesting apossible mechanism for the incomplete response to the CCR5 antagonist.

In one aspect, the present invention provides an in vivo method foridentifying a candidate compound that down regulate expression of CCR5and/or one or more of its ligand in tumor cells overexpressingendogenous or virally transduced oncogene selected from the groupconsisting of NeuT, Ha-Ras, c-Src and combinations thereof. In oneembodiment, the method comprises (a) administering a candidate compoundto an animal model of the tumor cells; (b) and measuring the level ofCCR5 RNA or protein expression in the animal model, wherein if the levelof CCR5 RNA or protein expression in the animal model is decreasedcompared to the level of CCR5 RNA or protein expression in untreatedanimal model, then the candidate compound is identified as a compoundthat down regulates expression of CCR5 and/or one or more of its ligandin tumor cells overexpressing endogenous or virally transduced oncogeneselected from the group consisting of NeuT, Ha-Ras, c-Src andcombinations thereof. In one embodiment, the tumor cells comprisemammalian prostate cancer cells. In one embodiment, tumor cells comprisemammalian prostate cancer cell line having at least one or more of a setof primary mammalian epithelial cells which have been infected with aretroviral vector carrying an oncogene selected from the groupconsisting of NeuT, Ha-Ras, c-Src and combinations thereof.

An exemplary mammalian prostate cancer cell lines suitable for use inthe present invention include, but are not limited to, mammalianprostate cancer cell lines disclosed in U.S. Provisional PatentApplication No. 61/646,586, filed May 14, 2012, whose contents areincorporated by reference herein in their entirety. In one embodiment,the mammalian prostate cancer cell line comprises at least one or moreof a set of primary mammalian epithelial cells which have been infectedwith a retroviral vector carrying an oncogene. In one embodiment, theoncogene is selected from the group consisting of c-Myc, Ha-Ras, NeuT,c-Src and combinations thereof. In one embodiment of the mammalianprostate cancer cell line, an oncogene selected from the groupconsisting of c-Myc, Ha-Ras, NeuT, c-Src and combinations thereof isexpressed. The mammalian prostate cancer cell line can include anysuitable mammalian cell, including primary murine epithelial cells. Insome embodiments, the primary mammalian epithelial cells are derivedfrom any immune competent mammal. In one embodiment, the primarymammalian epithelial cells are derived from an immune competent mammalselected from the group consisting of rodents, including rats and mice.

In some embodiments, the suitable animal model of cancer comprises animmune competent mammal implanted with a cancer cell line transformedwith one or more of a set of oncogenes selected from the groupconsisting of c-Myc, Ha-Ras, NeuT, c-Src and combinations thereof. Inone embodiment, the animal model of cancer is an immunocompetenttransgenic mouse created using the mammalian prostate cancer cell lineof the present invention develops a prostate tumor capable of producinga detectable molecular genetic signature based on an expression level ofone or more of a set of oncogenes selected from the group consisting ofc-Myc, Ha-Ras, NeuT, c-Src and combinations thereof.

In some embodiments, the suitable animal model of cancer is produced byan in vitro method. In one embodiment, in vitro method for producing thesuitable animal model includes production of immortalized primarymammalian epithelial cells. In one embodiment, the method for the invitro production of immortalized primary mammalian epithelial cells,comprises infecting primary mammalian epithelial cells with a retroviralvector carrying an oncogene selected from the group consisting of c-Myc,Ha-Ras, NeuT, c-Src and combinations thereof to provide infected cells,wherein the primary mammalian epithelial cells are capable of beinginfected by said retroviral vector and under conditions whereby thec-Myc, Ha-Ras, NeuT, c-Src and combinations thereof are expressed insaid infected cells.

I. EXAMPLES

A. Materials and Methods

A.1. Breast Cancer Patients Data Set and Statistical Analysis

A microarray data set that was previously compiled (21) from the publicrepositories Gene Expression Omnibus (23) and ArrayExpress (24) was usedto evaluate CCR5 and CCL5 expression in the context of clinical samples.Samples in this data set were assigned to 5 canonical breast cancersubtypes, including luminal A, luminal B, normal-like, basal, and HER-2− overexpressing disease. The classification of microarray samples amongthese 5 subtypes was achieved by computing their correlation against anexpression profile centroid representative of each subtype and assigningsamples to the subtype with the highest corresponding correlationcoefficient (25). Samples with a maximum correlation coefficient below0.3 were considered unclassified. Analysis of CCL5 and CCR5 transcriptwas then conducted specifically among the luminal A, luminal B, basal,normal-like, and HER-2 subtypes. Differential expression of the averagedgene signature magnitude among these sample subsets was evaluated using2-tailed Student t test. Kaplan-Meier analysis was used to evaluatesurvival trends within the sample subsets. Scatter plots of CCL5 versusCCR5 samples were also generated to observe coregulation patternsspecific to each subtype. For these scatter plots, gene profiles weremedian-centered and scaled to unitary SD.

A.2. Cell Lines and Cell Culture

MDA-MB-231, MCF-7, and Hs578T cells were maintained in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% FBS. SUM-159 cells(kindly provided by Dr. Stephen Ethier, Wayne State University, Detroit,Mich.) were maintained in Ham's F-12 supplemented with 4 μg/mL ofinsulin, 1 μg/mL of hydrocortisone, and 5% FBS. Oncogene-transformedderivatives of MCF-10A cells (MCF10A-NeuT, MCF10A-Src, and MCF10A-Ras;ref 26) were maintained in DMEM:Ham's F-12 (50 of 50) supplemented with4 mg/mL of insulin, 10 ng/mL of EGF, and 1 mg/mL of hydrocortisone. Atotal of 100 μg/mL of each penicillin and streptomycin were included inall media. Cells were cultured in 5% CO₂ at 37° C. For in vitrotreatments, maraviroc was dissolved in dimethyl sulfoxide (DMSO) anddiluted in culture medium. The final concentration of DMSO in treatedand control cultures was 0.5%. Vicriviroc was dissolved in culturemedium.

A.3. Fluorescence-Activated Cell-Sorting Analysis

Cell labeling and fluorescence-activated cell-sorting (FACS) analysisfor CCR5 were based on prior publications (27) with minor modifications.Before labeling, the cells were blocked with normal mouse IgG (1 of 100)and purified rat anti-mouse Fcγ III/II receptor antibody (1 of 100;Pharmingen) for 30 minutes and then incubated with allophycocyanin(APC)-labeled CCRS antibody (R&D Systems). All experiments wereconducted at 4° C. Sample analysis was conducted on FACSCalibur flowcytometer (BD Biosciences). These data were analyzed with FlowJosoftware (Tree Star, Inc.).

A.4. Invasion Assay

The 3-dimensional invasion assay was conducted as previously reported(12). Briefly, 100 μL of 1.67 mg/mL Rat Tail collagen type I (BDBiosciences) was pipetted into the top chamber of a 24-well 8 μm poreTranswell (Corning). The Transwell was incubated at 37° C. overnight toallow the collagen to solidify. A total of 30,000 cells were then seededon the bottom of the Transwell membrane and allowed to attach.Serum-free growth medium was placed into the bottom chamber, whereas 15ng/mL CCL5 or 5% FBS was used as a chemoattractant in the medium of theupper chamber. The cells were then chemoattracted across the filterthrough the collagen above for 3 days. Cells were fixed in 4%formaldehyde, permeabilized with 0.2% Triton-X in PBS, and then stainedwith 40 μg/mL propidium iodide (PI) for 2 hours. Fluorescence wasanalyzed by confocal z-sections (one section every 20 μm) at ×10magnification from the bottom of the filter using a Zeiss LSM 510 Metainverted confocal microscope at the Kimmel Cancer Center BioimagingFacility.

A.5. Intracellular Calcium Assay

Calcium responses induced either by CCL5 or FBS in human cancer celllines were monitored under fluorescence confocal microscope aspreviously reported (28). Briefly, breast cancer cells were seeded in4-well labtek chambers (Nunc) at 10⁴ cells/cm² and incubated for 1 day.

After 12-hour starvation, cells were labeled by incubating them with 2mmol/L Fluo-4-AM (Molecular Probes) in HBSS for 30 minutes, washedtwice, and incubated for additional 30 minutes before imaging under themicroscope. Time-lapse images were collected using a Zeiss LSM 510 Metainverted confocal microscope with the incubator at 37° C. Relativeintracellular Ca²⁺ concentration was determined by the changes influorescent intensity (FI) of Fluo-4-AM upon the addition of CCL5 (60ng/mL) or FBS (5%) and was calculated as (FI_(t)−FI₀)/FI₀.

A.6. MTT Assay

The MTT assay is a colorimetric assay for measuring the activity ofcellular enzymes that reduce the tetrazolium dye,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellowtetrazole (MTT), to its insoluble formazan, giving a purple color. Theseassay measures cellular metabolic activity via NAD(P)H-dependentcellular oxidoreductase enzymes and may, under defined conditions,reflect the number of viable cells (cell proliferation). Tetrazolium dyeassay can also be used to measure cytotoxicity (loss of viable cells) orcytostatic activity (shift from proliferative to resting status) ofpotential medicinal agents and toxic materials. MTT Assay usually donein the dark because MTT reagent is sensitive to light.

The effects of CCR5 antagonists on cell viability and proliferation ratewere estimated using the soluble tetrazolium salt MTT assay (29). MTT isreduced by the mitochondria of viable cells, and the amount of reducedformazan is proportional to the number of viable cells. After 72 hoursof exposure to the drugs, cells were incubated with 1 mg/mL of MTT for90 minutes. Then, the reduced (insoluble and colored) formazan wasdissolved in DMSO and measured spectrophotometrically at 570 nm. Theeffect of CCR5 over expression in breast cancer cell proliferation wasstudied in MDA-MB-231 cells transfected with full-length human CCR5subcloned into pcDNA3.1⁺/Zeo⁺ vector (kindly provided by Dr. EleanorFish, University of Toronto, Toronto, ON, Canada) and selected withZeocin (200μg/mL) as previously described (18). MTT assays wereconducted in sextuplicate using 96-well microplates.

A.7. Viral Cell Transduction

A lentiviral vector encoding firefly luciferase 2 (Luc2)-eGFP fusionprotein was a generous gift from Dr. Sanjiv S. Gambhir (School ofMedicine, Stanford University, Stanford, Calif.; ref. 30). Lentiviruspropagation was conducted following the protocol described by Zahler andcolleagues (31). Breast cancer cell lines were transduced at amultiplicity of infection of 20 in the presence of 8 mg/mL polybrene(Sigma) for 24 hours (30, 31).

A.B. Experimental Metastasis Assay And Bioluminescence Imaging

MB-MDA-231 cells expressing Luc2-eGFP (called MDA.pFLUG for the rest ofthe article) were detached with a nonenzymatic cell dissociation buffer(4 mmol/L EDTA in Ca²⁺ and Mg²⁻-free PBS), resuspended in Dulbecco's PBSwithout Ca²⁺ and Mg²⁺ and immediately injected into the tail vein of8-week-old, female nonobese diabetic/severe combined immunodeficient(NOD/SCID) mice (NCI, Bethesda Md.). Each mouse received 10 ⁶ cells.Mice were treated by oral gavage with maraviroc (8 mg/kg every 12 hours)or vehicle (5% DMSO in acidified water; ref. 32). Treatment was startedimmediately after injection or 10 days later for the experimentsanalyzing the proliferation of established metastasis. For in vivobioluminescence imaging (BLI), mice were given an intraperitoneal (i.p.)injection with 200 μL of d-luciferin (30 mg/mL). Mice were anesthetizedwith isoflurane (2% in 1 L/min oxygen), and bioluminescence images wereacquired 10 to 15 minutes after d-luciferin injection using the IVIS XRsystem (Caliper Life Sciences). Acquisition times ranged from 10 seconds(for later time points) to 5 minutes (for early time points). Data areexpressed as total photon flux and were analyzed using Living Image 3.0software (Caliper Life Sciences). For ex vivo BLI, d-luciferin wasdiluted in PBS to a final concentration of 300 μg/mL and used to soakfreshly isolated lungs for 2 to 3 minutes before imaging. Some lungswere stained with India ink, as previously reported (33), or processedand stained with hematoxylin and eosin to corroborate the presence ofpulmonary tumors. For homing assays, mice were euthanized 24 hours afterthe intravenous injection of MDA.pFULG cells. Lungs were perfused withPBS, fixed with freshly prepared formaldehyde (4% in PBS), and frozen inoptimum cutting temperature (Sakura Finetek). Cryosections (10 μm) werecounterstained with 4′,6-diamidino-2-phenylindole analyzed by confocalmicroscopy. Animal experiments were approved by the Thomas JeffersonUniversity's Institutional Animal Care and Use Committee.

A.9. Reagents and Antibodies

CCL5 (catalog no. 278-RN) and anti-CCR5 APC antibody (catalog no.FAB1802A) were purchased from R&D Systems. A rabbit anti-human CCR5polyclonal antibody (GenScipt; catalog no. A00979) was used forimmunohistochemical staining. Rat tail collagen type I was purchasedfrom BD Biosciences. Vicriviroc and maraviroc were obtained from SelleckChemicals. Luciferin was obtained from Gold Biotechnology.

B. Results

B.1. Active CCL5/CCR5 Signaling In Basal Breast Cancer

To examine the relative abundance of CCL5 and its receptor CCR5 bygenetic subtype, interrogation was conducted within a combinedmicroarray database comprising 2,254 human breast cancer samples from 27independent studies (21). The relative abundances of CCL5 and CCRS weresignificantly increased in the basal and HER-2 subtypes compared withthe normal-like, luminal A and luminal B subtypes (FIG. 1A). Theincreased expression of CCL5 and CCRS correlated positively inindividual breast cancer samples and the correlation was highlysignificant in the basal and HER-2 subtypes (FIG. 1B). The proportion ofpatients with a CCL5/CCR5-positive signature was more than 58% in thebasal and HER-2 subtypes (FIG. 1C). In agreement with previous reports,clinical information of the cases in this database showed that theprobability to develop metastasis is increased in the basal, luminal B,and HER-2 subtypes (FIG. 1D).

To determine the gene expression signaling pathway associated withenrichment of CCR5 and CCL5, GSEA analysis using KEGG and GO wasconducted of these tumor samples (FIG. 7A). These studies showedenrichment for gene expression of pathways including lymphocyteactivation, Janus-activated kinase (JAK)-STAT signaling, and Toll-likereceptor activation (FIG. 7A). The receptors for CCL5 include CCR1 andCCR3. Increased expression of CCL5 associated with increased CCR1, butnot CCR3, in the basal and HER-2 genetic tumor type (FIGS. 7B and 7C).In ER-negative patients receiving chemotherapy, there was aninsignificant trend toward reduced metastasis-free survival andrelapse-free survival in the increased CCR5 population, compared withthe population with reduced CCR5 expression (FIG. 7D). A comparison ofexpression levels for CCL5 versus CCR5, CCR1, and CCR3, comparing normalbreast with breast cancer showed increased correlation between receptorand ligand expression levels in tumors compared with healthy breasttissue (FIG. 8A).

B.2. Ccl5 Promotes Breast Cancer Ca²⁺ Signaling and Cellular Invasion

Three human breast cancer cell lines with a basal phenotype andmolecular signature: MDA-MB-231, Hs578T, and SUM-159 (34-37) were usedas models in the studies described herein. Analysis of CCR5 expressionby FACS showed that a small subpopulation of cells were positive for thereceptor in all 3 cell lines (FIG. 2A for MDA-MB-231 and FIGS. 9A and 9Cfor Hs578T and SUM-159). Because CCR5 activation induces calcium flux(38, 39), the activation of calcium signaling was assessed by CCL5.Addition of CCL5 to the cultures induced immediate calcium fluxes in asubpopulation of cells (FIG. 2B for MDA-MB-231 and FIGS. 9B and 9D forHs578T and SUM-159), providing evidence that CCR5 is functional in basalbreast cancer cells. As a positive control, the same cultures wereexposed to 5% FBS (40). Calcium flux, assessed by relative fluorescenceintensity, increased in more than 95% of the cells after FBS addition(FIG. 2B and FIGS. 9B and 9D). To further distinguish CCL5 -dependentsignaling, SUM159 cells were stably transduced with a CCR5 expressionvector and the Ca⁺² response to CCL5 versus FBS was conducted (FIGS. 10Cand 10D vs. 10E and 10F). CCR5 induced Ca⁺² signaling in theCCR5-overexpressing cells, whereas both lines responded similarly to FBSinduced Ca⁺² activation (FIGS. 10A-10F).

Next, the effect of CCR5 activation on breast cancer cell invasion wasassessed using 3D migration assays. CCL5 induced invasion of the basalMDA-MB-231, Hs578T, SUM-159 but not the luminal MCF-7 cells (FIGS. 2Cand D). CCL5 promoted invasion of MCF-10A cells engineered to expresseither NeuT, H-Ras, or c-Src oncogenes, compared with MCF10Avector-transduced cells (FIG. 2E and F), suggesting that CCL5responsiveness may be acquired during transformation and requiresspecific cooperative oncogenic signals. The finding that CCL5 inducedcellular invasion led us to examine the migratory capacity of CCR5⁺cells versus that of CCR5⁻ cells. Within the same SUM-159 breast cancercell line, CCR5⁺ cells showed an approximately 40-fold greater cellularinvasiveness (FIG. 2G and H), indicating that the expression of CCR5correlates with a proinvasive phenotype.

B.3. CCR5 Antagonists Block Breast Cancer Calcium Signaling and CellInvasion

The importance of CCR5 in HIV infection led to the development ofdifferent drugs that target this receptor. Therefore, examination ofwhether the CCR5 antagonists maraviroc and vicriviroc were capable ofblocking the CCL5/CCR5 signaling in basal breast cancer cells wereconducted. Both CCR5 antagonists blocked CCL5 -induced calciummobilization. In MDA-MB-231 cells, maraviroc and vicriviroc inhibitedcalcium responses by 65% and 90%, respectively (FIG. 3A and 3B). Similarobservations were made with both drugs in Hs578T cells (FIG. 3C and D),indicating that CCR5 expressed in different basal breast cancer cells issensitive to pharmacologic inhibition.

To evaluate the functional relevance of CCR5 in cellular migration andinvasion, the effects of maraviroc and vicriviroc were tested in 3Dinvasion assays. Using 2 different cell lines, it was found that bothCCR5 antagonists inhibited FBS-induced breast cancer cell invasion atthe clinically relevant concentration of 100 nmol/L (FIGS. 4A-4D). Thus,the proinvasive effect of CCR5 can be abrogated by using specificantagonists.

B.4. CCR5 Inhibition Blocks Breast Cancer Metastasis In Vivo

In view of the finding that CCR5 inhibition by CCR5 antagonists reducedcalcium signaling and cell invasion, the in vivo effect of maraviroc onlung metastasis was determined. MDA-MB-231 cells transduced within theLuc2-eGFP lentiviral vector (MDA.pFULG cells) were used in anexperimental metastasis model. The Luc2 gene is a codon-optimizedversion of Luc and cells expressing this reporter were 10 to 100 timesbrighter than the unmodified Luc gene (30). After injection of MDA.pFULGcells into the tail vein of mice, noninvasive BLI enabled the earlydetection of breast cancer metastasis (41). Weekly BLI was conducted for5 weeks and the radiance antemortem was used as a surrogate measurementof tumor burden. Mice treated with maraviroc (8 mg/kg twice daily)showed a significant reduction in both the number and the size ofpulmonary metastases compared with vehicle-treated mice (FIGS. 5A and B,FIGS. 11A and 11B). To avoid the possibility that metastases were missedbecause of inappropriate imaging, ex vivo imaging, India ink staining(FIG. 5C), and histology (FIG. 5E) of the lungs were conducted.Histologic analysis corroborated that tumor burden corresponds tobioluminescence, as previously shown (30). Metastatic tumors were stilldetectable in 50% of the maraviroc-treated mice, but their mean size wasreduced by 65% (FIGS. 5D and F). Interestingly, analysis of CCR5expression in lungs from control mice showed an 8-fold enrichment of theCCR5⁺fraction (FIG. 12). Collectively, these results provide evidencethat CCR5 antagonists reduce breast cancer metastasis in vivo.

B.5. CCR5 Antagonist Impairs Lung Colonization but Not CellProliferation or Tumor Growth

It was determined whether the reduction in metastatic tumors bymaraviroc involved changes in cellular proliferation and/or target organcolonization. The effect of CCR5 inhibition on cell viability andproliferation both in vitro and in vivo was analyzed. Maraviroc orvicriviroc treatment of MDA-MB-231 cells for 48 hours did not affect theMTT reduction, which was used as a surrogate measurement of cancer cellnumber (FIG. 6A). In agreement, over expression of CCR5 in MDA-MB-231cells did not modify their proliferation rate compared with cellstransfected with the empty vector (FIGS. 6B and 6C). Finally, maraviroctreatment of mice with established pulmonary metastasis did not modifytumor growth (FIGS. 6D and 6E), indicating that CCR5 activation does notpromote the proliferation of basal breast cancer cells in vitro nor inthe pulmonary microenvironment of immunocompromised mice.

On a different in vivo experiment, the effect of maraviroc on breastcancer cell homing to lungs was examined. To reach a steady-stateconcentration in plasma and tissues, mice were given 10 administrationsof maraviroc (twice a day for 5 days) before the intravenous injectionof MDA.pFULG cells (FIG. 6F). Inoculation of equal numbers of MDA.pFULGcells in control and treated groups was corroborated by BLI immediatelyafter injection. Maraviroc reduced the number of eGFP⁻ cells in thelungs by 40% (FIGS. 6G and 6H), suggesting that the in vivoantimetastatic effect of maraviroc is caused by a reduction in thenumber of cancer cells that colonize the target organ from thecirculation.

C. Discussion

The current studies show for the first time that: (i) enrichment ofCCL5/CCR5 expression occurs in patients with basal and Her2 positivegenetic subtypes of breast cancer; (ii) oncogenic transformation ofimmortalized human breast cells by distinct oncogenes induces CCLSresponsiveness; and (iii) maraviroc, an FDA-approved drug for thetreatment of CCR5 -trophic HIV infection, reduce metastatic tumor burdenin vivo.

Previous studies showed that CCL5 levels are elevated in breast primaryand metastatic tumors (9-11), suggesting a role of CCL5 in theacquisition of malignancy. The present disclosure show that increasedexpression of CCL5 and CCR5 are associated and that CCL5/CCR5 expressionlevels are different among the different genetic subtypes of breastcancer. Increased expression of CCL5 and CCR5 is found in the basal andHER-2 subtypes. In agreement, increased CCL5 expression has been foundpredominantly in ER-negative patients (42). Increased CCL5 alsocorrelated with increased CCR1 in basal and Her2 genetic subtypes ofbreast cancer. A trend toward reduced metastasis-free survival andrelapse-free survival was observed among the CCR5-overexpressing tumorsin patients who received chemotherapy.

Given the aggressive clinical behavior of basal breast cancer and thelack of targeted therapies for it, the importance of the CCL5/CCR5 axisin invasion and metastasis was evaluated in the human breast cancer celllines MDA-MB-231, Hs578T and SUM-159. These cell lines reflect theclinicopathologic features of the basal subtype of breast cancer(including the lack of HER-2, ER, and progesterone receptor), abasal-like molecular signature, the activation of specific signalingpathways (e.g., hypoxic or EGF receptor responses) and over expressionof epithelial-mesenchymal transition proteins (FN, VIM, and matrixmetalloproteinase 2; refs. 34-37). Only a small fraction of cells withinthe cell lines used in this study expressed CCR5 as evaluated by FACSanalysis. Our findings are consistent with studies by Muller andcolleagues who showed CCR5 expression in MDA-MB-231 by quantitativereal-time PCR (8). These studies confirmed the expression of CCR5 inMDA-MB-23 cells by reverse transcriptase PCR and showed the presence ofthe CCR5 protein by FACS analysis (FIGS. 13A-13D), and showed that CCR5immunohistochemical staining was localized primarily to the breastcancer epithelial cell, compared with normal breast tissue (FIGS.14A-14D).

The results herein show that CCL5 activates calcium flux in basal-likehuman breast cancer cells, as previously described in cells of theimmune system (39, 43) and CCR5 -transfected cells (27, 44, 45). Byusing the selective CCR5 -antagonists maraviroc and vicriviroc (bothwith IC₅₀ below 30 nmol/L; refs. 44, 45), it was shown that CCL5-activated signaling is mediated by CCR5. However, the fraction of CCL5-responsive cells (10% and 12% for MDA-MB-231 and Hs578T cells,respectively) is higher than the percentage of CCR5-expressing cellsdetermined by FACS. This may be due to the greater sensitivity of theCa²⁺ activation assays compared with the sensitivity of analysis byFACS. In addition, CCL5-induced calcium redistribution is not completelyblocked by CCR5 antagonists. This may be caused by the expression ofother receptors to CCL5, namely CCR1 and CCR3. CCR5 has been identifiedas the main CCL5 receptor in MDA-MB-231 cells (13) and CCR1 and CCR3transcripts are absent in both MDA-MB-231 or Hs578T cell lines (8) andbreast tumor samples (11). CCR1 and CCR3 were able to be detected byFACS (FIGS. 13A-13D), suggesting a possible mechanism for the incompleteresponse to the CCR5 antagonist.

It was observed that the subpopulation of CCR5⁺ cells displayedincreased invasiveness, indicating that CCR5 favors cell migration andinvasion in basal-like breast cancer cells. The failure of luminal-likeMCF-7 cells to respond to CCL5 is in agreement with previouspublications (12). These studies also showed that CCR5 inhibition witheither maraviroc or vicriviroc reduced in vitro FBS-induced breastcancer cellular invasion without affecting cellular viability. Thefinding that CCR5 antagonists block FBS-induced invasion is novel andsuggested that CCR5 activation contribute to the production ofmetastasis in vivo where different chemotactic and growth signals arepresent. The mechanisms involved in CCR5 regulation of FBS-activatedinvasiveness are uncharacterized but they may include heterodimerizationand ligand affinity regulation of other GPCRs (46), or thetransactivation of growth factor receptor (47) or integrin-mediatedsignaling (48), as described in noncancerous cells.

The in vivo antimetastatic effect of maraviroc was shown by injectingMDA.pFULG cells into the circulation of immunodeficient mice andtreating them with clinically relevant doses of the drug. In humans,oral doses of 300 mg produce an average C_(max) of 1,200 nmol/L (49),whereas in mice 16 mg/kg produce an average C_(max) of 1,045 nmol/L(32). Because the drug is taken twice a day in the clinical setting, 16mg/kg/d divided into 2 doses and administered during the experimentsdescribed herein. Maraviroc significantly reduced the pulmonary tumorburden. Although it has been proposed that pharmacologic CCR5 inhibitionmay be beneficial for patients with breast cancer, to our knowledge thisis the first study showing that systemic administration of a CCR5antagonist reduces metastatic colonization of basal breast cancer cells.

The antimetastatic effect of maraviroc is not caused by alterations ingrowth of established metastasis. CCR5 activation by CCL5 drivesproliferation in CCR5 -transfected MCF-7 breast cancer cells (18) andprostate cancer cells (50), but this study and others (13) showed thatthe CCL5/CCR5 axis does not play a role in cell proliferation orsurvival in the basal-like MDA-MB-231 cells. Furthermore, inhibition ofCCR5 surface expression through a dominant-negative form of CCR5(CCR5Δ32) in MDA-MB-231 cells does not change in vivo proliferation orapoptotic response (17). On the other hand, it was found that maravirocreduced lung colonization by MDA.pFULG cancer cells. This result isconsistent with previous studies in which inhibition of CCR5 expressionwithin breast cancer cells or administration of anti-CCL5 neutralizingantibody to tumor-bearing mice reduced the enhanced metastaticcapability induced by coinjection of mesenchymal stem cells (MSC; ref13). The authors identified cancer cell extravasation as the crucialmetastatic step affected by CCL5/CCR5 inhibition (13). Together, thesedata support a role for CCR5 antagonists in blocking the ability ofbasal breast cancer cells to reach the metastatic sites instead ofinhibiting their proliferation or survival after arrival. Blocking thehoming of cancer cells to metastatic sites is a desirable characteristicin a true antimetastatic drug (51). Therefore, CCR5 antagonists may beuseful as adjuvant therapy for breast basal tumors with CCR5 overexpression or other tumor types where CCR5 promotes metastasis, such asprostate cancer (50) or gastric cancer (52).

It will be appreciated by those skilled in the art that changes could bemade to the exemplary embodiments shown and described above withoutdeparting from the broad inventive concept thereof. It is understood,therefore, that this invention is not limited to the exemplaryembodiments shown and described, but it is intended to covermodifications within the spirit and scope of the present invention asdefined by the claims. For example, specific features of the exemplaryembodiments may or may not be part of the claimed invention and featuresof the disclosed embodiments may be combined. Unless specifically setforth herein, the terms “a”, “an” and “the” are not limited to oneelement but instead should be read as meaning “at least one”.

It is to be understood that at least some of the figures anddescriptions of the invention have been simplified to focus on elementsthat are relevant for a clear understanding of the invention, whileeliminating, for purposes of clarity, other elements that those ofordinary skill in the art will appreciate may also comprise a portion ofthe invention. However, because such elements are well known in the art,and because they do not necessarily facilitate a better understanding ofthe invention, a description of such elements is not provided herein.

Further, to the extent that the method does not rely on the particularorder of steps set forth herein, the particular order of the stepsshould not be construed as limitation on the claims. The claims directedto the method of the present invention should not be limited to theperformance of their steps in the order written, and one skilled in theart can readily appreciate that the steps may be varied and still remainwithin the spirit and scope of the present invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

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1-14. (canceled)
 15. A method of preventing metastasis of breast cancerin a subject having breast cancer, wherein the subject is at risk ofdeveloping metastasis of the breast cancer, the method comprising:administering to the subject who is at risk of developing metastasis ofthe breast cancer a therapeutically effective amount of a CCR5antagonist to prevent the metastasis of the breast cancer.
 16. Themethod of claim 15, wherein the CCR5 antagonist is selected from thegroup consisting of4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamideor a pharmaceutically acceptable salt thereof and(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)pheny]ethyl]-3-methylpiperazi-n-1-yl]-4-methylpiperidin-1-yl]methanoneor a pharmaceutically acceptable salt thereof.
 17. The method of claim15, wherein the breast cancer is basal breast cancer.
 18. The method ofclaim 15, wherein said CCR5 antagonist is selected from the groupconsisting of:4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide;N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide;N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide;N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide;N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarboxamide;and pharmaceutically acceptable salts or solvates thereof.
 19. Themethod of claim 15, wherein said CCR5 antagonist is selected from thegroup consisting of:N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide;1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide;2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;N-{(1S)-3-[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex-3-yl]-1-phenylpropyl}cyclobutanecarboxamide;2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-1,2,4-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide;N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide;N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;Methyl3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;N-{(1S)-3-[3-[3-endo(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide1-Acetyl-N-{(1S)-3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;1-Acetyl-N-{(1S)-3-[3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1]-1-phenylpropyl}-3-pyrrolidinecarboxamide;1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;1-Acetyl-N-{(1S)-3-[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;1-Acetyl-N-{(1S)-3-[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N(1S)-3-[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;1-Acetyl-N-{(1S)-3-[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;(2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;(2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]1-1-phenylpropyl}-3-azetidinecarboxamide;1-Methyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;2-Methoxy-N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;N-{(1S)-3-[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;3-Ethyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;3-Ethyl-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;N-{(1S)-3[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide;1-Acetyl-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-methoxyacetamide;N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;N{−1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methoxypropanamide;2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;and pharmaceutically acceptable salts thereof.
 20. The method of claim15, wherein the method prevents the metastasis of the breast cancer toone or more organs selected from the group consisting of liver, brain,bladder, lung, adrenal gland, kidney, bone and combinations thereof 21.The method of claim 15, wherein the CCR5 antagonist is administeredconcomitantly or concurrently with an additional therapeutic.
 22. Themethod of claim 15, further comprising identifying the subject withbreast cancer as being at risk of developing metastasis of the breastcancer prior to the administration of the CCR5 antagonist, theidentifying comprising: (a) obtaining a biological sample from thesubject having breast cancer; (b) measuring CCR5 level of expressionand/or level of expression of at least one of CCR5 ligands in thebiological sample, wherein if the measured CCR5 level of expressionand/or the level of expression of at least one of CCR5 ligandsdetermined in step (b) is increased compared to CCR5 level of expressionand/or level of expression of at least one of CCR5 ligands in a normalcontrol breast tissue sample, then the subject is identified at risk fordeveloping metastasis of the breast cancer.
 23. The method of claim 22,wherein measuring CCR5 level of expression and/or level of expression ofat least one of CCR5 ligands in the biological sample comprisesperforming a an RNA-based assay selected from the group consistingreverse transcription polymerase chain reaction or a microarray assay.24. The method of claim 22, wherein measuring CCR5 level of expressionand/or level of expression of at least one of CCR5 ligands in thebiological sample comprises performing an immunoassay selected from thegroup consisting of immunohistochemical staining or fluorescenceactivated cell sorting.